Survivin and Cancer
Survivin is overexpressed in a large portion of human cancers. (Altieri, D. C. (2003) Validating survivin as a cancer therapeutic target. Nat Rev Cancer 3: 46-54; Satoh et al. (2001) Expression of survivin is correlated with cancer cell apoptosis and is involved in the development of human pancreatic duct cell tumors. Cancer 92:271-278). Elevated Survivin levels frequently correlate with poor prognosis. Survivin appears to have two distinct biological roles. First, Survivin exhibits anti-apoptotic activity under certain experimental conditions (Li et al. (1999) Pleiotropic cell-division defects and apoptosis induced by interference with survivin function. Nat Cell Biol 1:461-466; Li et al. (1998) Control of apoptosis and mitotic spindle checkpoint by survivin. Nature 396:580-584). Indeed, Survivin was initially identified as a member of the Inhibitor of Apoptosis (IAP) family. (Id.). Although Survivin may not function by directly binding and inhibiting caspase, as initially thought, the current data suggest that Survivin antagonizes apoptosis by acting upstream of effector caspases (Dohi et al. (2004) Mitochondrial survivin inhibits apoptosis and promotes tumorigenesis. J. Clin Invest 114: 1117-1127; Song et al. (2003) Direct interaction between survivin and Smac/DIABLO is essential for the anti-apoptotic activity of survivin during taxol-induced apoptosis. J. Biol Chem 278:23130-23140). Activation of the checkpoint kinase Chk2 by DNA damage stimulates a rapid discharge of the mitochondrial pool of Survivin in the cytosol. (Ghosh et al. (2006) Activated checkpoint kinase 2 provides a survival signal for tumor cells. Cancer Res 66:11476-11579). Thus, Survivin may also be involved in modulation of DNA-damage-induced apoptosis.
Secondly, more recent studies indicate that Survivin is involved in chromosome segregation during mitosis. Survivin exists in immunologically distinct pools localized in various subcellular compartments, including kinetochores and microtubules. (Fortugno et al. (2002) Survivin exists in immunochemically distinct subcellular pools and is involved in spindle microtubule function. J. Cell Sci 115:575-585). Of note, Survivin is a component of the chromosomal passenger protein complex, which also include Aurora kinase B, INCENP, TD-60, and Borealin. This Survivin-containing protein ensemble contributes to spindle assembly by nucleating microtubules around mitotic chromosomes. (Sampath et al. (2004) The chromosomal passenger complex is required for chromatin-induced microtubule stabilization and spindle assembly. Cell 118:187-202). Survivin is localized to the kinetochore following chromosome condensation and becomes relocated to the midbody after chromosome segregation and until cytokinesis (Carvalho et al. (2003) Survivin is required for stable checkpoint activation in taxol-treated HeLa cells. J. Cell Sci 116:2987-2988; Temme et al. (2003) Localization, dynamics, and function of survivin revealed by expression of functional survivinDsRed fusion proteins in the living cell. Mol Biol Cell 14:78-92; Uren et al. (2000) Survivin and the inner centromere protein INCENP show similar cell-cycle localization and gene knockout phenotype. Curr Biol 10:1319-1328). It has been established that Survivin plays an essential role in modulating the spindle checkpoint control, a biological mechanism that ensures accurate chromosome segregation during mitosis and thus prevents aneuploidy (Musacchio and Hardwick (2002) The spindle checkpoint: structural insights into dynamic signaling. Nat Rev Mol Cell Biol 3:731-741). It is postulated that overexpression of Survivin may corrupt the spindle checkpoint control and thereby contribute to chromosome instability and aneuploidy.
Survivin and the Mitotic Checkpoint
During mitosis, the spindle checkpoint monitors the integrity of the kinetochore-microtubule attachment and ensure that cells do not enter anaphase until all the chromosomes form solid attachments with microtubules (Id.). Disruption of this checkpoint is associated with loss of chromosomes or premature exit from mitosis and, consequently, aneuploidy (Kops et al. (2005) On the road to cancer: aneuploidy and the mitotic checkpoint. Nat Rev Cancer 5:773-785). Chromosome separation is controlled through a cascade of signaling molecules, including Mad2, Cdc20 and the APC/C protein complex. A body of evidence substantiates the role of Survivin and the other chromosome passenger proteins in regulation of the spindle checkpoint. Depletion of Survivin in mammalian cells causes abnormal chromosome alignment and high levels of aneuploidy (Lens et al. (2003) Survivin is required for a sustained spindle checkpoint arrest in response to lack of tension. Embo J. 22:2934-2947; Li et al. (1999) Pleiotropic cell-division defects and apoptosis induced by interference with survivin function. Nat Cell Biol 1:461-466; Li et al. (1998) Control of apoptosis and mitotic spindle checkpoint by survivin. Nature 396:580-584). In particular, loss of Survivin function abrogates a spindle checkpoint and allows re-replication of DNA without completion of cell division (Lens et al. (2003) Survivin is required for a sustained spindle checkpoint arrest in response to lack of tension. Embo J. 22:2934-2947). It appears that Aurora B and Mad2 are not properly localized to the kinetochore in this condition (Id.). The essential role of Survivin in cell lines is consistent with the finding that genetic targeting of Survivin in mice leads to a lethal phenotype at the early embryonic stage (Uren et al. (2000) Survivin and the inner centromere protein INCENP show similar cell-cycle localization and gene knockout phenotype. Curr Biol 10:1319-1328).
As a component of the chromosomal passenger complex, Survivin contributes to chromatin-associated spindle formation (Sampath et al. (2004) The chromosomal passenger complex is required for chromatin-induced microtubule stabilization and spindle assembly. Cell 118:187-202). This process involves the activation of Aurora kinase B, which phosphorylates the mitotic centromere-associated kinesin (MCAK) and removes its microtubule depolymerizating activity (Tulu et al. (2006) Molecular requirements for kinetochore-associated microtubule-formation in mammalian cells. Curr Biol 16:536-541). Survivin is phosphorylated by Aurora kinase B (Beardmore et al. (2004) Survivin dynamics increases at centromeres during G2/M phase transition and is regulated by microtubule-attachment and Aurora B kinase activity. J. Cell Sci 117:4033-4042; Wheatley et al. (2004) Aurora B phosphorylation in vitro identifies a residue of survivin that is essential for its localization and binding to inner centromere protein (INCENP) in vivo. J. Biol Chem 279:5655-5660) and sequential modifications of Survivin through ubiquitination and deubiquitination affects its localization (Vong et al. (2005) Chromosome alignment and segregation regulated by ubiquitination of survivin. Science 310:1499-1504). In these events, Survivin may act as a sensor of kinetochore-microtubule interaction and constitute a component of the spindle-assembly checkpoint (Sandall et al. (2006) A Bir1-Sli15 complex connects centromeres to microtubules and is required to sense kinetochore tension. Cell 127 1179-1191).
A fraction of Survivin directly associates with polymerized microtubules during mitosis (Alfieri (2006) Targeted therapy by disabling crossroad signaling networks: the survivin paradigm. Mol Cancer Ther 5:478-482). In addition, the microtubule-bound Survivin can be phosphorylated by Cdc2 during mitosis, which stabilizes the Survivin protein. Microtubule-associated Survivin contributes to proper spindle formation, by modulating microtubule dynamics through formation of a larger complex. (O'Connor et al. (2000) Regulation of apoptosis at cell division by p34cdc2 phorphorylation of survivin. Proc Natl Acad Sci USA 97:13103-13107; Rosa et al. (2006) Survivin modulates microtubule thermodynamics and nucleation throughout the cell cycle. Mol Biol Cell 17:1483-1493).
Survivin and ErbB-Mediated Transformation
Aneuploidy is a hallmark in ErbB-transformed cells and is considered as a contributing factor to development of malignancy. Our previous studies indicate that activation of EGFR leads to upregulation of Survivin (Wang and Greene (2005) EGFR enhances Survivin expression through the phosphoinositide 3 (PI-3) kinase signaling pathway. Exp Mol Pathol 79:100-107). Notably, modulation of Survivin levels by EGFR is dependent on the PI-3 kinase pathway but not on the MAP kinase pathway. Similarly, the signaling events initiated by ErbB2 and ErbB3 also cause increase of Survivin levels (Asanuma et al. (2005) Survivin expression is regulated by coexpression of human epidermal growth factor receptor 2 and epidermal growth factor receptor via phosphatidylinositol 3-kinase/AKT signaling pathway in breast cancer cells. Cancer Res 65:11018-11025; Xia et al. (2006) Regulation of survivin by ErbB2 signaling: therapeutic implications for ErbB2-overexpressing breast cancers. Cancer Res 66:1640-1647). Collectively, these observations raise the possibility that ErbB receptors may cause transformation by directly affecting a molecule implicated in playing a dual role in apoptosis and chromosome stability.
Cancers appear to be adapted to the transformed phenotype and become dependent on the oncogenic mutations for their maintenance and survival. This phenomenon is more recently referred to as “oncogene addiction” but this term is actually implicit to the malignant phenotype. (Joners and Berns (2004) Oncogene addiction: sometimes a temporary slavery. Cancer cell 6:535-538). It is possible that certain tumors, which have overexpression of Survivin and ErbB molecules, may become “addicted” to the Survivin networks. The Survivin antagonists may thus offer therapeutic benefits by targeting cancers while limiting unwanted side effects.
Survivin as a Therapeutic Target for Cancer.
Overexpression of Survivin confers on cancer cells an increase of proliferative capacity and resistance to cell death, which translates into poor clinical prognosis. In contrast, Survivin is generally not in normal tissue (Altieri, D. C. (2003) Validating survivin as a cancer therapeutic target. Nat Rev Cancer 3: 46-54). Combined with its roles in apoptosis and chromosome segregation during mitosis, this makes Survivin an attractive target for cancer therapy (Altieri (2003) Validating survivin as a cancer therapeutic target. Nat Rev Cancer 3:46-54). First, previous studies showed that disruption of Survivin in tumor cells by a genetic approach leads to immediate cell-cycle arrest and spontaneous cell death. It is conceivable that molecules that target the Survivin-containing complex may inflict potent and efficient anti-cancer effects. Secondly, one of the widely explored approaches to develop cancer therapeutics is to induce aberrant mitosis in tumors, which usually leads to cell death. For example, taxol, a compound that blocks mitosis by stabilizing tubulin polymerization and interfering with formation of the mitotic spindle, has been used to treat various forms of human cancer. Because Survivin plays an essential role in mitosis, ablation of Survivin function causes cell mitotic arrest and cell death. Thus, small molecules that target Survivin function represent a new class of agents that interfere with both apoptosis and mitosis.
To date only a small number of Survivin targeting interventions have been developed. These include molecules designed or selected to limit Survivin expression, such as an antisense molecule (LY2181308 and SPC3042) and transcriptional repressors (YM155 and EM-1421). (Rodel et al. (2008) Survivin antisense oligonucleotide effectively radiosensitize colorectal cancer cells in both tissue culture and murine xenograft models. Int J. Radiat Oncol Biol Phys 71:247-255; Hansen et al. (2008) SPC 3042: a proapoptotic survivin inhibitor. Mol Cancer Ther. 7:2736-45; Nakahara et al. (2007) YM155, a novel small-molecule survivin suppressant, induces regression of established human hormone-refractory prostate tumor xenografts. Cancer Research 67:8014-8021; Lopez et al. (2007) The anticancer activity of the transcription inhibitor terameprocol (meso-tetra-O-methyl nordihydroguaiaretic acid) formulated for systemic administration. Anticancer Drugs 18:933-939). These approaches aim to perturb Survivin expression and thereby indirectly ablate Survivin functions. Survivin peptides have also been used in vaccination protocols to generate an immune response to cancer (Wobser et al. (2006) Complete remission of liver metastasis of pancreatic cancer under vaccination with HLA-A2 restricted peptide derived from the universal tumor antigen survivin. Cancer Immunol Immunother 55:1294-1298). The agents that directly disrupt the physical complex between Survivin and other network components are emerging but the success remains limited. In one example, Plescia et al. developed a class of compounds that act as the combined inhibitor of Survivin and HSP90 and exhibit promising anticancer activity in vivo (Plescia et al. (2005) Rational design of shepherdin, a novel anticancer agent. Cancer cell 7:457-468). In another example, a number of Survivin-binding molecules have been identified in a high-throughput, affinity-based screen (Wendt et al. (2007) Discovery of a novel small molecule binding site of human survivin. Bioorg Med Chem Lett 17:3122-3129). However, the biological activities of these compounds have not been documented.
Survivin Protein Structure.
Survivin is a protein of 142 amino acid residues. The structure of Survivin has been determined by both crystallography (Chantalat et al. (2000) Crystal structure of human survivin reveals a bow tie-shaped dimer with two unusual alpha-helical extensions. Mol Cell 6:183-189; Verdecia et al. (2000) Structure of the human anti-apoptotic protein survivin reveals a dimeric arrangement. Nat Struct Biol 7, 602-608) and by solution NMR methods (Sun et al. (2005) Solution structure of human survivin and its binding interface with Smac/Diablo. Biochemistry 44:11-17). The Survivin protein forms a homodimer that resembles a “bow tie”. The N-terminal region of Survivin contains a zinc-binding fold similar to the baculovirus IAP repeat (BIR) motif. This domain consists of a three-stranded β-sheet and four α-helices. The C-terminus of Survivin contains an extended α-helical coiled-coil domain. The solution NMR structure of a truncated version of the protein (1-120) unequivocally determined the dimer interface that was somewhat ambiguous in the crystal lattice (Id.).
The N-terminal region contains the structural features involved in dimerization and subcellular localization to the kinetochore and the midbody (Li and Ling (2006) Survivin study: an update of “what is the next wave”? J. Cell Physiol 208:476-486). In addition, mutations of the amino acid residues in this region affect the function of Survivin (Li et al. (1998) Control of apoptosis and mitotic spindle checkpoint by survivin. Nature 396:580-584). Notably, ubiquitination of Survivin in the N-terminal region modulates localization as well as degradation (Vong et al. (2005) Chromosome alignment and segregation regulated by ubiquitination of survivin. Science 310:1499-1504). A number of Survivin splice variants have been identified, such as Survivin-delta Ex3 lacking exon 3 and Survivin-2B retaining a part of intron 2 as a cryptic exon. Of note, the N-terminal region is shared by most of the variant forms (Li and Ling (2006) Survivin study: an update of “what is the next wave”? J. Cell Physiology 208:476-486; Noton et al. (2006) Molecular analysis of survivin isoforms: evidence that alternatively spliced variants do not play a role in mitosis. J. Biol Chem 281:1286-1295).
More recently, the structure of a Survivin-Borealin-INCENP Core Complex has been reported (Jeyaprakash et al. (2007) Structure of a Survivin-Borealin-INCENP core complex reveals how chromosomal passengers travel together. Cell 131:271-285). Borealin and INCENP associate with the c-terminal helical domain of Survivin to form a three-helical bundle of 1:1:1 stoichiometry. The interactions of the core components are essential for central spindle and midbody localization of the complex. Both Survivin and Borealin bind to the N terminus of INCENP (corresponding to the amino acid residues 1-58 of human INCENP), which is sufficient for targeting to the centromere (Ainsztein et al. (1998) INCENP centromere and spindle targeting: identification of essential conserved motifs and involvement of heterochromatin HP1. J. Cell Biol 143:1763-1774). Of note, Survivin is required for targeting of the Chromosome passenger protein complex to the centromere. There remains a need for effective therapeutics to disrupt Survivin.
Small molecules can be screened in silico to fit into the cavities on the surface of Survivin protein adjacent to the site involved in dimerization. (Wendt et al. (2007) Discovery of a novel small molecule binding site of human survivin. Bioorganic and Medicinal Chemistry Letters 17:3122-3129). Protein-Protein interactions can be disrupted by small molecules that lodge proximal to the interaction site (Murali et al. (2005) Disabling TNF receptor signaling by induced conformational perturbation of tryptophan-107. Proc. Natl. Acad. Sci. 102: 10970-75). Lodging of the small molecule into a cavity that disables the dimerization interface may lead to conformational changes that disrupt the formation of the dimer. Using this strategy, termed as “Cavity induced allosteric modification” (see WO 00/01349), we have identified an allosteric site and developed a new class of Survivin targeting small molecules by virtual screening (Ewing et al. (2001) DOCK 4.0: search strategies for automated molecular docking of flexible molecule databases. Comput. Aided Mol. Des. 15:411-428) that exhibit the ability to inhibit cell proliferation, block mitosis and cause tumor regression.
The currently used anti-Survivin strategies include suppressing surviving expression by antisense, ribozyme, siRNA, or shRNA approaches or antagonizing Survivin function by dominant negative Survivin. (Rosa et al. (2006) Survivin modulates microtubule dynamics and nucleation throughout the cell cycle. Mol Biol Cell 17:1483-1493). Recently, a small peptide, shepherdin, that blocks the interaction of heat shock protein 90 with Survivin, has been developed. (Plescia et al. (2005) Rational design of shepherdin, a novel anticancer agent. Cancer cell 7:457-468).